Leucine Inhibits Nitric Oxide & Beneficial Effects of Cardio Training on Glucose Management. Plus: No Increase in Protein Synthesis W/ Protein After Eccentric Workouts?

Pre-, Post and Intra-workout supplements are a multi-million dollar business. But are the products at least half as effective as the shiny advertisements claim?

Let me guess, the actual reason you want to read this article is the shocking claim that everybody's darling, the "pro-anabolic amino acid" leucine may, in addition to having beneficial effects on the phosphorylation of mTOR and the subsequent increase in skeletal muscle protein synthesis, also have negative effects. Right? Ok, I will add a link that takes you right to my elaborations on two recent studies from the São Paulo University and the China Agricultural University and allows you to skip past the information about another recent study.

A study from the Department of Public Health at the Aarhus University and a study that raises the question, whether its results are indicate that eccentric training is potent enough to maximize the protein anabolic response to an extend that the additional provision of dietary protein will not lead to further increases in markers of protein synthesis.
Before we draw any conclusions, I guess we should first take a look at what Stine Klejs Rahbek and her colleagues actually did: To investigate the effects of leucine-rich whey protein hydrolysate and carbohydrate (WPH+CHO) versus isocaloric carbohydrate (CHO) supplementation on the Akt-mTOR and the AktFOXO signaling axis, during recovery from muscle-damaging exercise and to evaluate whether their hypothesis that WPH+CHO would accentuate signaling for protein synthesis and attenuate signaling for protein degradation, compared to isocaloric CHO, the researchers recruited twenty-four young healthy recreationally active men who had not participated in systematic resistance training or eccentric dominated activities for lower extremity muscles within 6 months prior to inclusion in the study.

The study itself was conducted in a double blinded, isocaloric placebo-controlled fashion in regards to dietary supplementation. Following inclusion, subjects were randomly allocated into either

• a whey protein hydrolysate+carbohydrate group (WPH+CHO, n = 12) or
• isocaloric carbohydrate placebo group (CHO, n = 12)

On the exercise trial day, subjects reported to the laboratory at 07.30 am in a fasted state. Before the eccentric exercise protocol was initiated, muscle soreness was evaluated using a visual analog scale, a blood sample was collected and knee extensor muscle contractile function was evaluated. Subsequently, a unilateral eccentric exercise protocol for was completed, lasting approximately 30 min (see below).

Figure 1: Timeline of interventions and measurements on the four study days are shown. A muscle biopsy was sampled 14 days prior to the exercise trial (i.e. to establish basal level). The protocol for days 1 and 2 was identical (Rahbeck. 2015).

Immediately after the exercise bout, the subjects ingested either a WPH+CHO or a CHO supplement, according to the group they were assigned, and then rested for 3 h. At 3-h post-exercise, a biopsy was obtained from both the exercise and the non-exercise control leg. Before leaving the laboratory, the subjects ingested the second drink (1.00 pm) and received a third drink to ingest 3 h later (4.00 pm).

"On days 1 and 2 (24 and 48 h following exercise, respectively), the subjects were instructed to ingest three supplements at absolute time points similar to day 0, with the fist drink always ingested after the functional tests and biopsy sampling. Biopsy sampling from both the ECC and the CON leg on days 1 and 2 were performed under conditions similar to the pre-exercise biopsy, i.e., the subjects fasted overnight and rested in the supine position for 45 min prior to biopsy sampling. The biopsy sampling on days 1 and 2 was timed to correspond to 24 and 48 h following exercise termination. Assessments on indices of muscle damage (muscle force, muscle soreness and plasma muscle creatine kinase) were repeated at 24, 48, 72, 96, and 168 h after overnight fasting." (Rahbeck. 2015)

All Subjects were instructed to refrain from physical activity such as exercise, stair case walking and other types of strenuous activity in the hours/ days between post-exercise assessments of indices of muscle damage to ensure that all measured effects made actually reflected the effects of the exercise + supplementation intervention.

Figure 2: Effects of eccentric training + supplementation on markers of protein anabolism (left, green) and catabolism (right, red) as measured in the trained leg (Rahbeck. 2015).

Speaking of effects: If you look at the data in Figure 2, you will realize that the eccentric training led to significant decreases in muscle force (by 23–27 % at 24 h post-exercise), which were followed by gradual, although not full recovery at 168 h post-exercise, with no differences between supplement groups. Furthermore, the phosphorylation of mTOR, p70S6K and rpS6 (=the activity of these protein anabolic proteins) increased and phosphorylation of FOXO1 and FOXO3 (=the activity of these catabolic proteins) decreased in the ECC leg, again with no differences between supplement groups.
If this is not your first visit to the SuppVersity you will yet know that these observations are not sufficient to warrant the previous voiced hypothesis that the exercise induced increase in protein synthesis in response to eccentric exercises realizes as long as you are training "hard enough", because mTOR, p-p70S6K & co are no reliable measure of the actual amount of protein that's transported into the muscle after the workout.

Don't freak out about the Rahbeck study, take a look at the latest studies leucine science!

Instead of freaking out that your protein supplements may be useless, you may thus rather discard this study as interesting, but inconclusive and take a closer look at the latest evidence that leucine, when consumed in excess and isolation could have side effects you may want to avoid.

• Leucine + endurance exercise - no perfect match? In their latest study, scientist from the São Paulo University were able to show that "leucine supplementation did not potentiate the effects of endurance training on protein turnover, and it also reduced its positive effects on glucose homeostasis" (Costa Junior. 2015) - in rodents.
  
  In view of the fact that the interactions between endurance exercise, leucine supplementation and glucose metabolism have not previously been studies, the scientists analyzed the effects of endurance exercise training plus leucine supplementation on protein turnover and glucose homeostasis in healthy mice.
  

  Figure 3: Changes in body composition (left), changes in the expression of the catabolic ubiquitin-proteasome protiens (middle) and effects on glucose disappearance index aka effective glucose uptake after the swimming workout (right | Costa Junior. 2015).

  The results you see in Figure 3 tell you three things: (a) Leucine did not have the previously described minor minor beneficial effects on body composition (increases in lean mass are compensated by increases in fat mass); (b) it did not ameliorate the protein turnover during exercise and (c) it did actively blunt the real world increase in glucose uptake after the endurance workout in spite of the fact that changes in marker proteins like AMPK (not shown)would suggest otherwise.
  
  Reason to panic? No, the effects are not pronounced enough. In view of the fact that the benefits of isolated leucine supplementation and "spiking" other supplements with extra leucine are totally overblown, anyway. This may be the final straw that brakes the leucine guzzling camel's neck for those of you who's primary goal is to use exercise to improve their glucose tolerance.
• Leucine an "anti-pump", "anti-heart health" supplement? While they are a bit removed from human in vivo studies, the claims made by Yang et al. in their latest paper in Amino Acids are a bit frightening.
  
  Based on the observation that increased concentrations of l-leucine in the plasma occur in obese humans and other animals with vascular dysfunction, the scientists argue that the unique inhibitory effect of leucine on NO synthesis from l-arginine in endothelial cells may be part of why the increase in serum BCAA that's brought about by a failure to metabolize the branch-chained amino acids in the obese could negatively modulate cardiovascular homeostasis in insulin resistance.
  

  Figure 4: This is how leucine messes with NO synthesis. It increases the production of glutamine:fructose- 6-phosphate aminotransferase (GFAT) which then shuts down NO synthesis (Yang. 2015).

  "Results of recent studies indicate that l-leucine is an activator of glutamine:fructose- 6-phosphate aminotransferase (GFAT), which is the fist and a rate-controlling enzyme in the synthesis of glucosamine (an inhibitor of endothelial NO synthesis). Through stimulating the mammalian target of rapamycin signaling pathway and thus protein synthesis, l-leucine may enhance GFAT protein expression, thereby inhibiting NO synthesis in endothelial cells" (Yang. 2015).

  Yang et al. propose that reducing circulating levels of l-leucine or endothelial GFAT activity may provide a potentially novel strategy for preventing and/or treating cardiovascular disease in obese and diabetic subjects and highlight:
  

  "Such means may include dietary supplementation with either α-ketoglutarate to enhance the catabolism of l-leucine in the small intestine and other tissues or with N-ethyll-glutamine to inhibit GFAT activity in endothelial cells" (Yang. 2015).

  If the scientists (reasonable) assumptions are accurate, anything that prevents the leucine-induced activation of GFAT, be it nutritional supplements or pharmaceutical drugs, may in fact contribute to improved cardiovascular function by enhancing vascular NO synthesis. For the average trainee that's not really relevant, but if you look at the composition of contemporary N.O. boosters this revelation may explain why the "old" NO Xplode with arginine, caffeine & co worked significantly better than its BCAA-laden successors. 

References:

• Asp, Sven, Jens R. Daugaard, and Erik A. Richter. "Eccentric exercise decreases glucose transporter GLUT4 protein in human skeletal muscle." The Journal of physiology 482.Pt 3 (1995): 705-712.
• Costa Junior, et al. "Leucine supplementation does not affect protein turnover and impairs the benefiial effects of endurance training on glucose homeostasis in healthy mice." Amino Acids (2015): Ahead of Print.
• Kirwan, J. P., et al. "Eccentric exercise induces transient insulin resistance in healthy individuals." J Appl Physiol 72.6 (1992): 2197-202.
• Rahbek, Stine Klejs, et al. "No differential effects of divergent isocaloric supplements on signaling for muscle protein turnover during recovery from muscle-damaging eccentric exercise." Amino Acids (2015): 1-12.
• Yang, Ying, et al. "l-Leucine and NO-mediated cardiovascular function." Amino acids (2015): 1-13.